Melatonin is a neuroprotective hormone with pleiotropic properties, many of which are modulated by the mitochondria. Melatonin is found in neuronal mitochondria. However, little is known about the synthesis or transport of melatonin in neurons. Our preliminary data suggest that melatonin is produced in neuronal mitochondria and the hypothesis of this project is that neuronal melatonin is synthesized in the mitochondrial matrix; that AANAT activity (the rate limiting enzyme in the melatonin synthesis pathway) in the mitochondria is regulated by phosphorylation and interaction with the 14-3-3 chaperone, and that melatonin synthesized in the mitochondria remains in the neuron where it acts locally to protect neurons from cellular stress. To test this hypothesis, we will propose a research design that will accomplish three Aims. Aim 1 will characterize the mitochondrial melatonin synthesis pathway, including localizing each of the four melatonin synthesis enzymes and determining how they are transported to subcellular membrane and organelles. In Aim 2 we will determine the mechanisms responsible for regulating melatonin synthesis in neurons. Aim 3 will focus on the intracellular transport of mitochondrially-produced melatonin. The data generated from this project will provide a novel foundation for understanding the protective action of melatonin on neurons and opens a new research avenue for mitochondrial melatonin pharmacology and biology.